CN106770161A - A kind of method for quick of food-borne pathogens - Google Patents
A kind of method for quick of food-borne pathogens Download PDFInfo
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- CN106770161A CN106770161A CN201611165348.1A CN201611165348A CN106770161A CN 106770161 A CN106770161 A CN 106770161A CN 201611165348 A CN201611165348 A CN 201611165348A CN 106770161 A CN106770161 A CN 106770161A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Abstract
A kind of method for quick of food-borne pathogens, step is：Nanometer human lymph node post added with food-borne pathogens reference culture bacterium solution is scanned, the linear ratio relation of its corresponding SERS and its characteristic peak, Raman peak intensity and corresponding pathogenic bacteria quantity is obtained；It is according to the Raman spectrum data storehouse for setting up each pathogenic bacteria type and quantity of correspondence with these data；Sample drop after sample pre-treatment is added on nanometer human lymph node post, scanning obtains its SERS；The SERS of sample is compared with Raman spectrum data database data, if having all characteristic peaks of certain pathogenic strain SERS in database in sample Raman spectrum, then judge sample in have the strain, otherwise then without；Retrieval is carried out with the SERS of sample and the spectroscopic data of various concentrations in database and matches to determine the quantity of the strain in sample.
Description
Technical field
The present invention relates to a kind of method for quick of food-borne pathogens, belong to food-borne pathogens detection technique neck
Domain.
Background technology
In the last few years, food security accident all over the world frequently occurred, wherein, the outburst rate of food origin disease is located always
In propradation, food origin disease has turned into a worldwide public health that is huge and constantly extending with food pollution
Problem.
Food origin disease is caused by food-borne pathogens more, therefore, how food-borne pathogens are detected rapidly and sensitively
Presence turned into control food security key point.
The food-borne pathogens being currently known include Gram-negative bacteria and gram-positive bacteria, wherein, Gram-negative
With Salmonella CMCC50041 and vibrio parahemolyticus ATCC17802 as representative, gram-positive bacteria increases Liszt to bacterium with single
Bacterium ATCC19115 is that representative is most dangerous.
The traditional detection method of food-borne pathogens will by being separately cultured, the step such as biochemical identification, there is operation
Complex steps, to take many, detection sensitivity low and easy the shortcomings of there is false negative, in the sudden public thing of food security of reply
On part, it is impossible to meet that quick, accurate, sensitive and specificity is high etc. to be required.
Although the Fast Detection Technique of some food origin diseases is there now have been, including such as micro- life of conventional context of detection
Immunomagnetic isolation technology in terms of the proprietary enzyme fast reaction technology of thing, hydrophobicity grid filter membrane law technology, immunology detection, exempt from
Epidemic disease diffusion technique, the electrical impedance technology of metabolism context of detection, microcalorimetry technology, ATP bioluminescence techniques, molecular biology
DNA probe technology, polymerase chain reaction technology of context of detection etc., but during these Fast Detection Techniques of application, before sample
Treatment is same extremely complex time-consuming, and experiment accuracy also suffers from the influence of staff's operative skill.
Chinese patent literature CN102115778A discloses a kind of to " the SERS mirror of food-borne pathogens
Other method ".Although the technical scheme carries out cluster analysis by using SERS detection technique, realizing to many
There is preferable, quick Detection results aspect to propose a solution to plant food-borne pathogens.
But, according to the present inventor to the further investigation using SERS detection technique, find above-mentioned skill
Still there is following defect in art method：
First, not only need the bacteria samples that will have been pre-processed with enhancing substrate mix vibration after can just carry out plus survey, and
And the consumption of the usage amount and the pathogenic bacteria that limit gold or silver nanoparticle colloidal sol matching quantitatively is needed when being vibrated；
Second, the method cleans bacterium using distilled water, it is contemplated that distilled water can shadow in the process with bacterium contact cleaning
Ring the osmotic pressure of bacterium, cause bacterial death, so as to influence to detect the degree of accuracy of viable bacteria；
Third, the method carries out cluster analysis after bacterium spectrum is obtained to it, follow-up unknown bacteria samples are then using poly-
Alanysis combination bacterium spectral signature peak makes a distinction to bacterium；This analysis process still seems comparatively laborious；
Fourth, the sample that above-mentioned analysis method is used is pure bacterium sample, without for the inspection of bacterium present in actual sample
Survey and propose, therefore there is a certain distance in its specific aim.
In view of above-mentioned analysis, inventors believe that provide it is a kind of it is practical, can either be quick, and with more correct
The method for detecting various food-borne pathogens, is still association area problem in the urgent need to address.
The content of the invention
The purpose of the present invention：It is intended that overcoming the deficiencies in the prior art, there is provided a kind of quick detection of food-borne pathogens
Method, to the purpose for reaching simplified detecting step, shortening detection time.
It is that, up to above-mentioned purpose, the present invention provides following technical scheme：
The method for quick of this food-borne pathogens, comprises the following steps：
A) Raman spectrometer is used, the condition of scanning as power output 30mw, a length of 785nm of probing wave, sweep time is set
It is 5s, to added with food-borne pathogens：Salmonella CMCC50041, vibrio parahemolyticus ATCC17802, single increasing Liszt
The nanometer human lymph node post of bacterium ATCC19115 reference culture bacterium solutions is scanned, it is determined that having 731cm-1、798cm-1、1646cm-1
Characteristic peak is Salmonella CMCC50041 SERSs, with 573cm-1、898cm-1、1149cm-1Feature
Peak is vibrio parahemolyticus ATCC17802 SERSs, with 566cm-1、895cm-1、1414cm-1Characteristic peak
Be Listeria monocytogenes ATCC19115 SERSs；
B) by the SERS of above-mentioned pathogenic bacteria reference culture and each reference culture surface-enhanced Raman light
The linear ratio relation for composing Raman peak intensity and corresponding reference culture quantity, it is established that each reference culture type and quantity of correspondence
Raman spectrum data storehouse；
C) pre-treatment is carried out to sample, is added drop-wise on nanometer human lymph node post after obtaining sample liquid, it is identical with step a)
The condition of scanning it is scanned, obtain sample SERS and each SERS draw
Graceful peak intensity degrees of data；
D) the Raman spectrum data database data that the SERS of above-mentioned sample is set up with step b) is entered
Row matching is compared, if a certain SERS in sample has certain pathogenic bacteria reference culture table in database
Face enhancing Raman spectrum in all characteristic peaks, then judge sample in have the strain, otherwise then without；With sample
SERS and the spectroscopic data of lane database various concentrations carry out retrieval and match to determine the bacterium in sample
The quantity planted.
Described reference culture bacterium solution be after reference culture activation through 36-38 DEG C Liquid Culture 8-9 hours to logarithmic phase, so
Nutrient solution is cleaned and resulting bacterial suspension after resuspension operation with physiological saline afterwards.
Described sample liquid is by sample and physiological saline volume ratio 1 by weight:After the ratio mixing of 8-12, it is put into
In homogenizing bag, patted 2-5 minutes in homogenizer；Then its supernatant is taken, at centrifugation under the conditions of 600-1000rpm, 3-5 DEG C
Reason 5-10 minutes, then supernatant is transferred in centrifuge tube obtains the thick liquid of sample, the thick liquid of sample is cleaned with physiological saline again and
Bacterial suspension is obtained after being operated through resuspension.
In aforesaid operations, described its bulking value specific concentration of physiological saline is 0.85%.
In aforesaid operations, described resuspension operation is in 3500- by the thick liquid of nutrient solution or sample after cleaning
Centrifugal treating 3-8 minutes under the conditions of 4500rpm, 3-5 DEG C,
Compared with prior art, the present invention is provided Methods for Fast Detection of Foodborne Pathogenic Bacteria its advantage and significantly enter
Step is：
1) present invention utilizes SERS technology, swept by food-borne pathogens reference culture bacterium solution
Retouch, its SERS and its characteristic peak and raman scattering intensity data are obtained, according to this as sample strain and bacterium amount
SERS contrast standard, thus reaching need to be only by comparing in sample SERS
The no characteristic peak occurred in contrast standard, it is possible to whether judge in sample containing the pathogenic bacteria corresponding to characteristic peak, enter
One step can also match survey according to the Raman spectrum of the pathogenic bacteria in sample with the retrieval of concentration known spectrum in database
Go out the quantity of the pathogenic bacteria in sample.
2) detection method that the present invention is provided is establishing food-borne pathogens reference culture SERS figure
After database, to sample pathogenic bacteria detection only need the simple process of 25 minutes or so is first carried out to sample, again through 5
The Raman spectroscopy and comparing of or so minute can be completed, and need that detection knot could be obtained within 7-8 hours than prior art
Fruit not only simplify the detecting step of food-borne pathogens, detection time also be highly shortened, for food security has been provided
Effect technical guarantee；
3) the food-borne pathogens detection method that the present invention the is provided spy such as have simple to operate, quick, accurate, sensitivity high
Point, thus great application value.
Brief description of the drawings
Fig. 1 is the SERS figure of food-borne pathogens reference culture.
In figure：
Its Surface-Enhanced Raman Scattering Characteristics peak of Salmonella CMCC50041 is 731cm-1、798cm-1、1646cm-1；
Its Surface-Enhanced Raman Scattering Characteristics peak of vibrio parahemolyticus ATCC17802 is 573cm-1、898cm-1、1149cm-1；
Its Surface-Enhanced Raman Scattering Characteristics peak of Listeria monocytogenes ATCC19115 is 566cm-1、895cm-1、1414cm-1。
Specific embodiment
SERS (surface-enhanced Raman spectroscopy, SERS) is a kind of Gao Ling
Quick Raman detection technology, signal enhancing is mediated with metal Nano structure, has resolution ratio higher compared to normal Raman spectroscopy
And sensitivity, its principle is the electromagnetic field of sample surfaces or near surface in some precious metal surfaces or colloidal sol of special preparation
Enhancing, the phenomenon for causing the Raman scattering signal for producing binding molecule to greatly enhance than normal Raman scattered signal occurs, this
Signal enhancing is planted up to 6~14 orders of magnitude.
The signal interference of same substance in solution can be prevented effectively from due to SERS, be selected with high sensitivity and surface
Selecting property, can obtain high-quality surface Raman signal, realize up to 1014 times of unimolecule Raman signal enhancing, therefore, low
The context of detection very advantageous of concentration analysis thing, can clearly show the characteristic peak of sample, for qualitative analysis is provided
Basis.
Raman spectrum depends on the inelastic scattering of exciting light and intermolecular vibration to produce finger-print, with quick inspection
Various chemistry, the ability of biological substance are surveyed, specific molecule to be checked is differentiated with this, real-time detection can be carried out.
Because the Ramam effect of water is very weak so that Raman spectrum is highly effective to the biology sample detection in water environment.Cause
This, there is this feature of different vibrating Raman spectrum using different food-borne pathogens, by simple sample pre-treatments, just
The detection of food-borne pathogens can be in minutes completed, compensate for prior art is carried out to food borne pathogens in food
During detection, cumbersome this shortcoming of preceding processing operations for a long time must be carried out, simplify detecting step, shorten detection time.
A kind of method for quick of the food-borne pathogens for providing the present invention in conjunction with the embodiments is made further specifically
It is bright.
Embodiment
A) SERS of reference culture is obtained
Respectively by Salmonella CMCC50041, vibrio parahemolyticus ATCC17802, Listeria monocytogenes ATCC19115
Reference culture activation, then Liquid Culture 8~9 hours, to logarithmic phase, then uses weight respectively under the conditions of 36~38 DEG C respectively
Volume ratio is that 0.85% physiological saline cleans bacterium solution 3 times, each draws 1mL bacterium solutions, carry out 4000rpm, under the conditions of 4 DEG C from
The resuspension operation of heart 5min, then, then draws the μ L of bacterium solution 4 of different strain, is added separately on nanometer human lymph node post, in Raman
Spectrometer power output obtains the surface enhanced of each reference culture under the conditions of 30mw, a length of 785nm of probing wave, to scan 5 seconds
Raman spectrum.
Shown in food-borne pathogens reference culture SERS figure as shown in Figure 1：
Its Surface-Enhanced Raman Scattering Characteristics peak of Salmonella CMCC50041 is 731cm-1、798cm-1、1646cm-1；
Its Surface-Enhanced Raman Scattering Characteristics peak of vibrio parahemolyticus ATCC17802 is 573cm-1、898cm-1、1149cm-1；
Its Surface-Enhanced Raman Scattering Characteristics peak of Listeria monocytogenes ATCC19115 is 566cm-1、895cm-1、1414cm-1。
B) the Raman spectrum data storehouse of correspondence reference culture is set up
The SERS of above-mentioned each pathogenic bacteria reference culture is included into its characteristic peak data and each standard bacteria
The linear ratio relation of strain SERS Raman peak intensity and corresponding reference culture quantity, it is established that corresponding each standard
The Raman spectrum data storehouse of bacterial strain type and quantity；
Can also be according further to the intensity of Raman peaks in the corresponding SERS of reference culture, combined standard
The data such as the concentration of bacterial strain bacterium solution, the quantity being added on nanometer human lymph node post, phase is drawn by the database for retrieving well-established
The bacterial strain quantity of pass；With this, it is established that the Raman spectrum data storehouse of correspondence reference culture.
C) SERS of sample is obtained
Take the sample for being polluted by food-borne pathogens some, volume ratio is 1 by weight:10 ratio adds body
Product specific concentration is 0.85% physiological saline, is put into homogenizing bag after mixing, and 2min is patted with homogenizer, then takes 2mL supernatants
Liquid, is centrifuged treatment 8min under the conditions of 800rpm, 4 DEG C, and supernatant is transferred into 1.5mL centrifuge tubes after abandoning precipitation
It is interior, obtain the thick liquid of sample；Then the thick liquid of sample is cleaned 3 times with the physiological saline that bulking value concentration is 0.85%, then is carried out
4000rpm, the resuspension operation that treatment 5min is centrifuged under the conditions of 4 DEG C, its suspension are sample liquid；
Then, 4 μ L sample liquid are drawn, is added drop-wise on nanometer human lymph node post, with embodiment step a) the identical conditions of scanning
It is scanned, the SERS of sample is obtained.
D) type and quantity of food-borne pathogens in sample are determined
The Raman spectrum data that the sample SERS of above-mentioned acquisition and embodiment step b) are set up
Whether data in storehouse carry out matching comparing, with the surface-enhanced Raman in sample with certain pathogenic strain in database
Whether spectral signature peak judges to have the strain in sample：
If including 731cm in the SERS in sample-1、798cm-1、1646cm-1These peak values,
It is determined that containing Salmonella CMCC50041 in sample；
If including 573cm in the SERS in sample-1、898cm-1、1149cm-1；These peaks
Value, it is possible to judge to contain vibrio parahemolyticus ATCC17802 in sample；
If including 566cm in the SERS in sample-1、895cm-1、1414cm-1These peak values,
It is determined that containing Listeria monocytogenes ATCC19115 in sample；
Retrieved with the various concentrations spectroscopic data of the Raman spectrum of sample bacterial strain corresponding with database, compared
It is right, so that it may to draw the quantity of the strain in sample.
It is visible in sum：
By the scanning to food-borne pathogens reference culture bacterium solution obtain its SERS and in this, as
The contrast standard of sample SERS, need to only carry out the simple process of 25 minutes or so to sample, then
Through the simple process of 5 minutes or so, then Raman spectroscopy and Data Comparison through 5 minutes or so, so that it may complete to food-borne
The detection of pathogenic bacteria type and quantity.
Obviously, the 7-8 hour obtainable testing result of ability is needed relative to present existing detection technique, using we
Rule can be completed in the time of 30 minutes or so.Not only enormously simplify the detecting step of food-borne pathogens, moreover it is possible to and it is big
Shorten detection time in ground；To ensure that food security provides new reliable detection means, with extensive promotional value.
Finally, it is necessary to which explanation is：
The above is only used for help and understands technical scheme, it is impossible to be interpreted as the limit to the scope of the present invention
System；The non-intrinsically safe modifications and adaptations that those skilled in the art are made according to the above that the present invention is provided, belong to the present invention
Required protection domain.
Claims (6)
1. a kind of method for quick of food-borne pathogens, it is characterised in that comprise the following steps：
A) Raman spectrometer is used, the condition of scanning is set as power output 30mw, a length of 785nm of probing wave, sweep time are 5s,
To added with food-borne pathogens：Salmonella CMCC50041, vibrio parahemolyticus ATCC17802, Listeria monocytogenes
The nanometer human lymph node post of ATCC19115 reference culture bacterium solutions is scanned, it is determined that having 731cm-1、798cm-1、1646cm-1It is special
Levy peak is Salmonella CMCC50041 SERSs, with 573cm-1、898cm-1、1149cm-1Characteristic peak
Be vibrio parahemolyticus ATCC17802 SERSs, with 566cm-1、895cm-1、1414cm-1Characteristic peak
It is Listeria monocytogenes ATCC19115 SERSs；
B) SERS of above-mentioned pathogenic bacteria reference culture and each reference culture SERS are drawn
The linear ratio relation of graceful peak intensity and corresponding reference culture quantity, it is established that the Raman of each reference culture type and quantity of correspondence
Spectra database；
C) pre-treatment is carried out to sample, is added drop-wise on nanometer human lymph node post after obtaining sample liquid, swept with step a) identicals
Retouch condition to be scanned it, obtain the SERS of sample and the feature of each SERS
Peak data；
D) the Raman spectrum data database data that the SERS of above-mentioned sample and step b) set up is carried out
With comparison, if certain pathogenic bacteria reference culture surface increases during a certain SERS in sample has database
All characteristic peaks in strong Raman spectrum, then judge there is the strain in sample, on the contrary then without；With the surface of sample
Enhancing Raman spectrum and the spectroscopic data of various concentrations in database carry out retrieval and match to determine the strain in sample
Quantity.
2. the method for quick of a kind of food-borne pathogens according to claim 1, it is characterised in that：Described sample
Liquid is by sample and physiological saline volume ratio 1 by weight:After the ratio mixing of 8-12, it is put into homogenizing bag, in homogenizer
Middle beating 2-5 minutes；Then its supernatant is taken, centrifugal treating 5-10 minutes under the conditions of 600-1000rpm, 3-5 DEG C, then will be upper
Clear liquid obtains the thick liquid of sample in being transferred to centrifuge tube, by the thick liquid of sample again with physiological saline clean and through resuspension operate after
To bacterial suspension.
3. the method for quick of a kind of food-borne pathogens according to claim 1, it is characterised in that：Described standard
Bacterial strain bacterium solution be after reference culture activation through 36-38 DEG C Liquid Culture 8-9 hour to logarithmic phase, then will culture with physiological saline
Liquid is cleaned and resulting bacterial suspension after resuspension operation.
4. the method for quick of a kind of food-borne pathogens according to claim 1, it is characterised in that：Described sample
Liquid is by sample and physiological saline volume ratio 1 by weight:After the ratio mixing of 8-12, it is put into homogenizing bag, in homogenizer
Middle beating 2-5 minutes；Then its supernatant is taken, centrifugal treating 5-10 minutes under the conditions of 600-1000rpm, 3-5 DEG C, then will be upper
Clear liquid obtains the thick liquid of sample in being transferred to centrifuge tube, by the thick liquid of sample again with physiological saline clean and through resuspension operate after
To bacterial suspension.
5. the method for quick of a kind of food-borne pathogens according to Claims 2 or 3, it is characterised in that：Described
Physiological saline, its bulking value specific concentration is 0.85%.
6. the method for quick of a kind of food-borne pathogens according to Claims 2 or 3, it is characterised in that：Described
Resuspension operation is that centrifugation 3-8 divides under the conditions of 3500-4500rpm, 3-5 DEG C by the thick liquid of nutrient solution or sample after cleaning
Clock.
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| CN110146486A (en) * | 2019-05-31 | 2019-08-20 | 西安工程大学 | A kind of active pathogen in-situ detection method based on Ta@Ag micro-nano interface |
| CN111398241A (en) * | 2020-01-15 | 2020-07-10 | 武汉市农业科学院 | High-flux SERS detection method for food-borne pathogenic bacteria |
| CN111912931A (en) * | 2020-08-10 | 2020-11-10 | 燕山大学 | TLC-SERS quantitative modeling method based on quaternion principal component analysis |
| CN112033949A (en) * | 2020-09-04 | 2020-12-04 | 中国农业大学 | Method for rapidly detecting aquatic product spoilage bacteria by SERS (surface enhanced Raman scattering) biosensor |
| CN112098389A (en) * | 2020-08-31 | 2020-12-18 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN113567418A (en) * | 2021-08-06 | 2021-10-29 | 海南微氪生物科技股份有限公司 | Method for detecting pathogenic microorganisms by fusing spectrum technology |
| CN113702353A (en) * | 2021-08-25 | 2021-11-26 | 苏州四灵纳米生物科技有限公司 | Bacterial liquid cleaning method and system for Raman detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110146486A (en) * | 2019-05-31 | 2019-08-20 | 西安工程大学 | A kind of active pathogen in-situ detection method based on Ta@Ag micro-nano interface |
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| CN111398241A (en) * | 2020-01-15 | 2020-07-10 | 武汉市农业科学院 | High-flux SERS detection method for food-borne pathogenic bacteria |
| CN111912931A (en) * | 2020-08-10 | 2020-11-10 | 燕山大学 | TLC-SERS quantitative modeling method based on quaternion principal component analysis |
| CN112098389A (en) * | 2020-08-31 | 2020-12-18 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN112098389B (en) * | 2020-08-31 | 2022-04-22 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN112033949A (en) * | 2020-09-04 | 2020-12-04 | 中国农业大学 | Method for rapidly detecting aquatic product spoilage bacteria by SERS (surface enhanced Raman scattering) biosensor |
| CN113567418A (en) * | 2021-08-06 | 2021-10-29 | 海南微氪生物科技股份有限公司 | Method for detecting pathogenic microorganisms by fusing spectrum technology |
| CN113702353A (en) * | 2021-08-25 | 2021-11-26 | 苏州四灵纳米生物科技有限公司 | Bacterial liquid cleaning method and system for Raman detection |
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Application publication date: 20170531 |